An immunoassay using an antigen-antibody reaction, a DNA hybridization assay or the like has conventionally been used for microanalysis of biological samples. In such analyses, an antibody or DNA is required to be labeled with the labeling agents. The labeling agents generally used are those modified with fluorescence, radioisotope or enzyme, which enable highly sensitive detection.
Radioisotope labeling, although highly sensitive, has some disadvantages involving risks in storage, use and disposal. Enzyme labeling also has some defects. Because of a high molecular weight of an enzyme, the labeling stability and reproducibility may often be impaired by the external factors such as temperature. The activities of both enzyme and the labeled substance may often be reduced due to the bindings of the enzyme labeling agent to the substance to be labeled.
As fluorescent labeling methods, the labeling with organic fluorescent dye such as fluorescein, rhodamine and dansyl chloride has been known. However, it has a defect in that the fluorescence detection is likely to be disturbed to a large extent by the background noises due to the scattered excitation light and the fluorescence response derived from other coexisting substances within a sample, whereby making a highly sensitive measurement difficult.
The labeling with a rare-earth fluorescent complex has also been known as one of the fluorescent labeling methods. The rare-earth fluorescent complex has a long fluorescence lifetime (of from several tens to several hundreds of microseconds or longer compared with a common organic fluorescent substance of which fluorescent lifetime is several nanoseconds), a large Stokes shift and a sharp fluorescent peak, which enables the highly sensitive fluorescence measurement if used for a time-resolved fluorescence assay, by eliminating a short-life background noises derived from an excitation light or other coexisting substances. The time-resolved fluorescence assay with the use of the rare-earth fluorescent complex of the above described characteristics as a labeling agent has already been developed.
The 2,2′:6′,2″-terpyridine derivative has been reported as one of the rare-earth fluorescent complexes (See Patent Reference 1). The 2,2′:6′,2″-terpyridine derivative directed to form a complex with a radioactive metal in use for a radioactive reagent has also been reported (See Patent Reference 2).
Meanwhile, the present inventors have already developed chlorosulfonyl quadridentate beta-diketone labeling agents that can directly label a protein having an amino group and have investigated an application thereof to a time-resolved fluorescence assay (See Patent References 3 and 4). However, said labeling agent of a chlorosulfonylated tetradentate β-diketone type generally had a defect of poor solubility in water, whereby reduced the solubility of the labeled biomaterials and resulted in precipitation from the solution, particularly when the labeled biomaterials were small (for example, a nucleic-acid base of a low molecular weight having amino groups, and other organic compounds). The buffers usable were limited due to the insufficient chelating ability thereof, which was another defect.
The present inventors have developed a large number of labeling agents of rare-earth fluorescent complexes and studied the application thereof to the time-resolved fluorescence assay. The already developed N,N,N′,N′-{2,6-bis-(3′-aminomethyl-1′-pyrazolyl)-4-phenyl pyridine}-tetra-acetic acid (hereinafter abbreviated to as BPTA) was able to form a complex with terbium and europium and emit strong fluorescence but had a maximum excitation wavelength at 320 nm which was short. On the other hand, (2,2′,2″,2′″-{4′-{[(4,6-dichloro-1,3,5-triazine-2-yl)-amino]-biphenyl-4-yl}-2,2′:6′2″-terpyridine-6,6″-di-yl)-bis-(methylene nitro)tetra acetic acid} (hereinafter abbreviated to as DTBTA) was able to be excited at 340 nm or longer, but was able to form a fluorescent complex only with europium (See Patent Reference 5).
It is important for the fluorescent labeling agent to have the longer excitation wavelength in order to overcome instrumental limitation involved in optical elements such as a lens and a light source as well as minimize the effect to the samples, therefore the development of a rare-earth fluorescent complex with excitation wavelength of 340 nm or longer has been desired.
DNA probe method has widely been employed in DNA analysis. Both a standard sample and a test sample (analyte) labeled with two or more labeling agents are furnished to the same probe DNA, followed by quantitatively determines the amount of DNA in a test sample based on the results of competitive hybridization. For the method like this, the construction of a fluorescence emitting complex with a plurality of rare-earth ions is desired.    Patent Reference 1: JP3-500297/1991, A    Patent Reference 2: JP7-506667/1995, A    Patent Reference 3: JP9-241233/1997, A    Patent Reference 4: JP2000-111480, A    Patent Reference 5: WO2003/076938